An Immunogenic and Slow-Growing Cryptococcal Strain Induces a Chronic Granulomatous Infection in Murine Lungs

Abstract

Many successful pathogens cause latent infections, remaining dormant within the host for years but retaining the ability to reactivate to cause symptomatic disease. The human opportunistic fungal pathogen Cryptococcus neoformans establishes latent pulmonary infections in immunocompetent individuals upon inhalation from the environment. These latent infections are frequently characterized by granulomas, or foci of chronic inflammation, that contain dormant and persistent cryptococcal cells. Immunosuppression can cause these granulomas to break down and release fungal cells that proliferate, disseminate, and eventually cause lethal cryptococcosis. This course of fungal latency and reactivation is understudied due to limited models, as chronic pulmonary granulomas do not typically form in mouse cryptococcal infections. A loss-of-function mutation in the Cryptococcus-specific MAR1 gene was previously described to alter cell surface remodeling in response to host signals. Here, we demonstrate that the mar1Δ mutant strain persists long term in a murine inhalation model of cryptococcosis, inducing a chronic pulmonary granulomatous response. We find that murine infections with the mar1Δ mutant strain are characterized by reduced fungal burden, likely due to the low growth rate of the mar1Δ mutant strain at physiological temperature, and an altered host immune response, likely due to inability of the mar1Δ mutant strain to properly employ virulence factors. We propose that this combination of features in the mar1Δ mutant strain collectively promotes the induction of a more chronic inflammatory response and enables long-term fungal persistence within these granulomatous regions.

Keywords: Cryptococcus neoformans; GM-CSF; Titan cell; cell cycle defects; cell wall; granuloma; hypoxia.

FIG 1

Pulmonary granulomatous region formation in murine cryptococcal infections. (A) Dissected lungs of female C57BL/6 mice infected with 1 × 10⁵ cells of the WT strain or mar1Δ mutant strain were harvested at clinical endpoints corresponding to imminent mortality. Gross organ examination revealed large, well-circumscribed inflammatory regions (white arrowheads) in mar1Δ mutant strain-inoculated lungs compared to diffuse lung injury in WT strain-inoculated lungs. (Created with BioRender.com). (B to E) Lungs of female C57BL/6 mice inoculated with 1 × 10⁵ cells of the WT strain or the mar1Δ mutant strain sacrificed at the predetermined endpoints of 3 (B), 7 (C), 14 (D), and 40 (E) dpi were harvested for histopathological analyses. Hematoxylin and eosin staining was utilized to visualize microscopic lung pathology (fungal cells [yellow arrowheads], inset [yellow boxes]). The 5× scale bar (left) is 250 μm, and the 10× scale bar (right) is 50 μm. (F) Granulomatous region diameter (in micrometers) was measured using Fiji. Data are summarized from one slide from three mice per strain per time point. σ, standard deviation (micrometers). The gray box indicates no experimental subjects can be assessed at this time point.

FIG 2

Contributions of GM-CSF signaling to the pulmonary granulomatous response. (A to C) The lungs of female (n = 2) (shown) and male (n = 2) (not shown) Csf2rb^−/− mice inoculated with 1 × 10⁵ cells of the WT strain or the mar1Δ mutant strain sacrificed at the predetermined time points of 3 (A), 7 (B), and 14 (C) dpi were harvested for histopathological analyses. Hematoxylin and eosin staining was utilized to visualize microscopic lung pathology (fungal cells [yellow arrowheads], inset [yellow boxes]). The 5× scale bar (left) is 250 μm, and the 10× scale bar (right) is 50 μm. (D) Pulmonary fungal burden of female (n = 2) and male (n = 2) Csf2rb^−/− mice inoculated with 1 × 10⁵ cells of the WT strain or the mar1Δ mutant strain sacrificed at 3 dpi was measured by quantitative cultures. Error bars represent the standard error of the mean (SEM). Statistical significance was determined using Student's t test (*, P < 0.05).

FIG 3

Fungal burden throughout infection. Pulmonary fungal burden of female C57BL/6 mice (n = 15) inoculated with 1 × 10⁴ cells of the WT strain or the mar1Δ mutant strain was measured by quantitative cultures throughout infection: 3, 7, 14, and 21 dpi. Brain fungal burden of female C57BL/6 mice (n = 15) inoculated with 1 × 10⁴ cells of the WT strain or the mar1Δ mutant strain was measured by quantitative cultures at 14 and 21 dpi. Error bars represent the SEM. Statistical significance was determined using Student's t test (***, P < 0.001; ****, P < 0.0001; ns, not significant).

FIG 4

Pulmonary cytokine profile and leukocyte infiltrate associated with the granulomatous response. (A) Pulmonary cytokine responses of female C57BL/6 mice inoculated with 1 × 10⁴ cells of the WT strain or the mar1Δ mutant strain were measured using the Bio-Plex protein array system throughout infection: 1 (n = 15), 3 (n = 15), 7 (n = 10), 14 (n = 10), and 21 (n = 10) dpi. Error bars represent SEM. Statistical significance between strains at each time point was determined using two-way ANOVA (***, P < 0.001; ****, P < 0.0001; ns, not significant). Only a subset of data is shown; refer to Fig. S6 in the supplemental material for full analysis. (B) Pulmonary leukocyte infiltrates of female C57BL/6 mice inoculated with 1 × 10⁴ cells of the WT strain or the mar1Δ mutant strain were measured by flow cytometry throughout infection: 1, 3, 7, and 21 dpi. Data shown are the mean ± SEM of absolute cell numbers from three independent experiments (n = 3) performed using five mice per group per time point per experiment. Error bars represent SEM. Statistical significance between strains at each time point was determined using two-way ANOVA (*, P < 0.05; ***, P < 0.001; ns, not significant). Only a subset of data is shown; refer to Fig. S7 for full analysis. (C) Pulmonary macrophage activation of female C57BL/6 mice (n = 3) inoculated with 1 × 10⁴ cells of the WT strain or the mar1Δ mutant strain was measured by flow cytometry throughout infection: 7, 14, and 21 dpi. Inducible nitrogen oxide synthase (iNOS) was used as a marker for M1 macrophages, and arginase 1 (Arg1) was used as a marker for M2 macrophages. The percentages of total iNOS⁺ cells and Arg1⁺ cells are shown. Error bars represent the SEM. Log transformation was used to normally distribute the data for statistical analysis. Statistical significance between strains at each time point was determined using two-way ANOVA. AM, alveolar macrophage (CD45⁺ CD11b⁻); IM, interstitial macrophage (CD45⁺ CD11b⁺). (Created with BioRender.com).

FIG 5

Pathogenesis-relevant virulence factor phenotypes of the mar1Δ mutant strain. (A) Titan cell formation was induced in the WT strain, the mar1Δ mutant strain, and the mar1Δ + MAR1 complemented strain. Cells were pregrown in YNB medium at 30°C, and an OD₆₀₀ of 0.001 was transferred to 10% HI-FBS in PBS incubated at 5% CO₂ at 37°C for 96 h. Cells were imaged by DIC microscopy (Zeiss Axio Imager A1). Cell diameter was measured using Fiji, and cells with a diameter of >10 μm were considered Titan cells (red arrowheads). The number of Titan cells per 10,000 cells was calculated for each strain. A minimum of 400 cells were analyzed across three biological replicates (n = 3). Error bars represent the SEM. Statistical significance was determined using a one-way ANOVA (*, P < 0.05; ns, not significant). The 63× scale bar is 10 μm. (B) The WT strain, the mar1Δ mutant strain, and the cap59Δ mutant strain were incubated in YPD medium at 30°C and CO₂-independent medium (TC) at 37°C until saturation. Samples were subsequently fixed, mounted, dehydrated, and sputter coated. Samples were imaged with a Hitachi S-4700 scanning electron microscope to visualize capsule organization and elaboration.

FIG 6

Slow-growth phenotypes of the mar1Δ mutant strain. (A) Morphological defects were analyzed in the WT strain, the mar1Δ mutant strain, and the mar1Δ + MAR1 complemented strain through incubation in YPD medium at either 30°C or 37°C. Cells were imaged by DIC microscopy (Zeiss Axio Imager A1) and were subsequently visually inspected for morphological defects, such as elongated cells (red squares), wide bud necks (red arrowhead), and cytokinesis failure (red circle). The percentage of total cells displaying morphological defects was quantified for each strain at each temperature. A minimum of 500 cells were analyzed across three biological replicates (n = 3). Error bars represent the SEM. Log transformation was used to normally distribute the data for statistical analysis. Statistical significance between strains at each time point was determined using two-way ANOVA (*, P < 0.05; **, P < 0.01; ns, not significant). The 63× scale bar is 10 μm. (B) Growth of the WT strain, the mar1Δ mutant strain, and the mar1Δ + MAR1 complemented strain was assessed in YPD medium at 37°C. Growth was tracked for 40 h and was measured by absorbance at OD₆₀₀. The figure summarizes data across three biological replicates (n = 3). Error bars represent the SEM. (C) Hypoxia resistance was assessed by growth on YES medium supplemented with CoCl₂ (0.7 mM) and in a microaerophilic chamber. Serial dilutions of the WT strain, mar1Δ mutant strain, mar1Δ + MAR1 complemented strain, and sre1Δ mutant strain were spotted onto agar plates and incubated at 30°C. Results were compared to the same strains grown on YES medium under ambient air conditions. (D) The WT strain, mar1Δ mutant strain, and mar1Δ + MAR1 complemented strain were incubated on YES medium with and without CoCl₂ (0.7 mM) at 30°C under ambient air conditions. After 72 h of growth, cells were isolated and imaged by DIC microscopy (Zeiss Axio Imager A1). Cell diameter (in micrometers) was measured using Fiji. The average cell diameter was quantified for each strain under each condition. A minimum of 50 cells were analyzed across two biological replicates (n = 2). Error bars represent the SEM. Statistical significance was determined using a two-way ANOVA (*, P < 0.05; **, P < 0.01; ns, not significant). The 63× scale bar is 10 μm.